Microfluidic Devices for Cellular Sorting

ABSTRACT

Microfluidic devices for cell sorting or cell fractionation are disclosed. A microfluidic device can comprise one or more inlets, a first wall and a second wall, and two or more outlets. The first and second walls can be substantially planar to each other and the first wall having can have a plurality of ridges protruding from the first wall and defining a compression gap between the ridge and a surface of the second wall. The microfluidic device can also be a cell sorting device for sorting a plurality of cells based on one or more biophysical cellular properties including size, elasticity, viscosity, and/or viscoelasticity wherein the cells are subjected to one or more compressions due to the compression gap. Also disclosed are methods for cell sorting based on a variety of biophysical cellular properties.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/774,684 filed 9 May 2018, which is a § 371 of InternationalApplication No. PCT/US2016/061141 filed 9 Nov. 2016, which claims thebenefit of US Provisional Patent Application No. 62/252,709 filed 9 Nov.2015, the entire contents and substance of which are hereby incorporatedby reference as if fully set forth below.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numberCBET-0932510 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

Not Applicable

SEQUENCE LISTING

Not Applicable

STATEMENT REGARDING PRIOR DISCLOSURES BY THE INVENTOR OR A JOINTINVENTOR

Not Applicable

BACKGROUND OF THE DISCLOSURE 1. Field of the Disclosure

The various exemplary embodiments of the disclosure relate generally toprocesses, methods, and systems for cellular sorting. It is particularlyuseful for cell sorting based on a variety of biophysical properties andbiomechanical properties, including elasticity, viscosity, andviscoelasticity, and high purity cell sorting.

2. Background

In recent years, the links between disease states, cell function, andcell biophysical properties have been actively studied in a variety ofpathologies, including cancer, malaria, and sickle cell anemia. Althoughdetermining biophysical properties of cells is currently employed inclinical care, particularly differences in cell density and size,utilizing new biophysical markers in conjunction with existingbiochemical and biomolecular assays has the potential to greatlyincrease the sensitivity and specificity of detecting target cells.

Viscoelasticity is a biophysical property of cells that exhibits bothelastic (reaction to deformation) and viscous (reaction to the rate ofdeformation) properties after undergoing stress and deformation.Viscoelasticity is a very important property of cells undergoing anydynamic process, such as the deformation of blood cells duringcirculatory flow. For example, changes in the ability of blood cells todeform and relax can result in severe complications. Whereas healthyleukocytes and erythrocytes deform when passing through capillaries andrestore to their original shape after exit, highly viscous and stifferleukemia cells, caused from altered cytoplasmic composition and enlargednucleus, result in increased blood flow resistance. Variation inelasticity and viscosity of the red blood cells also affects themicrocirculatory blood flow, which results in diminished ability totransport oxygen in sickle cells patients. Therefore, sorting andenriching cells by viscoelastic properties has tremendous clinicalvalue.

Cell separation is also vital in several clinical diagnosticapplications such as detection of cancer and infectious diseases. Inhospitals and laboratories, cell separation is routinely carried out bycentrifugation, size exclusion, and cell sorters based on fluorescentsignals. The demand to obtain high-purity cell population at low costspurred growing interest in exploring alternative cell separationmethods based on microfluidics. For example, electric fields can be usedto separate cells in microfluidic channels based on cell surface chargedistribution. Magnetic fields can be used to separate cells withdifferent surface proteins that bind magnetic particles in microfluidicchannels. Also, acoustic fields can be used to separate cells ofdifferent sizes. In addition, research studies have shown that cellbiomechanical properties including size and stiffness are biomarkers ofdiseases such as cancer and malaria. The changes in cell biomechanicalproperties are attributed to the transformation of cellular structuressuch as the cytoskeleton and nucleus. Therefore, cell biomechanicalproperties are phenotypes that could potentially be used for clinicaldiagnostics and therapies. Several recent studies have used hydrodynamicforce and optical force to distinguish cell phenotypes based on cellmechanical properties.

Utilizing differences in cell biomechanical properties for diseasedetection would be greatly aided by means to sort cells biomechanically.Biomechanical properties are intrinsic to the cell and as such provide alabel-free approach to enrichment without the need to discover anddevelop biomolecular reagents to aid detection. In addition,microfluidic platforms enable continuous sample processing, improvesensitivity, and utilize small sample volume. Recently, severalmicrofluidic cell sorting approaches based on variations in cellbiomechanical properties have been demonstrated. However, the ultimatepurity of the separated cells is limited by the intrinsic variabilityand overlap of the biomechanical properties of different cell types,even if the average properties are substantially different. As a result,biomechanical approaches to cell enrichment are in general inferior tothe best results achieved by cell sorting methods based on antibodybinding of magnetic or fluorescent signals.

BRIEF SUMMARY OF THE DISCLOSURE

The various exemplary embodiments of the disclosure relate generally toprocesses, methods, and systems for cell sorting using microfluidicdevices

An exemplary embodiment of the disclosure can be a microfluidic devicecomprising a first wall and a second wall, the walls being substantiallyplanar to each other, and the first wall having a plurality of ridges.The plurality of ridges can be diagonally oriented with respect to acentral axis of the microfluidic device and each respective ridge of theplurality of ridges can be separated by a ridge spacing. Each ridge ofthe plurality of ridges can protrude normal to the first wall and definea compression gap between the ridge and a surface of the second wall.The compression gap can have a height that is about 4 to about 16microns (μm), about 5 to about 14 microns, or about 6 to about 11microns. Each ridge of the plurality of diagonally oriented ridges canform a ridge angle with respect to the central axis of the microfluidicdevice. The ridge angle can be about from about 20 to about 70degrees)(° ), about 30 degrees, about 45 degrees, or about 60 degrees.Additionally, the microfluidic device can comprise at least 7 ridges,but may comprise 7 to 21 ridges, or 14 ridges.

Each respective ridge of the plurality of ridges can be separated by aridge spacing. The ridge spacing can have a width that can be about 50to about 350 microns or about100 to about 300 microns. In some exemplaryembodiments, the width of the ridge spacing is about 100 to about 200microns and a ridge angle formed by at least one ridge with respect tothe central axis of the microfluidic device is about 30 degrees. In someexemplary embodiments, width of the ridge spacing is about 100 micronsor less and a ridge angle formed by at least one ridge with respect tothe central axis of the microfluidic device is about 45 degrees. In someexemplary embodiments, the width of the ridge spacing is about 200microns, a ridge angle formed by at least one ridge with respect to thecentral axis of the microfluidic device is about 30 degrees, and theplurality of ridges comprises 30 ridges.

The microfluidic device can comprise one or more inlets and two or moreoutlets. In some exemplary embodiments, the microfluidic device maycomprise three outlets. The microfluidic device can also comprise anexpansion region downstream from the plurality of ridges. At least oneoutlet of the microfluidic device can comprise a flow apportionmentregion, a flow balancing region, and a collection point. In someexemplary embodiments, at least one flow apportionment region can have adifferent size than at least a second flow apportionment region. In someexemplary embodiments, at least one flow balancing region can comprise aserpentine channel.

An exemplary embodiment of the disclosed microfluidic device can be acell sorting device for sorting a plurality of cells based on one ormore biophysical cellular properties including size, elasticity,viscosity, and/or viscoelasticity. The cell sorting device can comprisean inlet for flowing a cell medium comprising the plurality of cellsinto the device at a flow velocity, a plurality of outlets forcollecting sorted portions of the plurality of cells, and a top planarwall and a bottom planar wall. The top planar wall can comprise aplurality of ridges protruding normal to the top planar wall anddefining a compression gap between a surface of the bottom planar walland each ridge of the plurality of ridges. Additionally, each ridge ofthe plurality of ridges can be oriented diagonally with respect to acentral flow axis and each respective ridge of the plurality of ridgescan be separated by a diagonally oriented ridge spacing. Exemplaryembodiments of the cell sorting device can comprise some or all of theaspects discussed above with respect to the microfluidic device.

In addition to the inlet, the cell sorting device can also comprise oneor more sheath flow inlets. The sheath flow inlets can be used forflowing a sheath fluid into the cell sorting device and provide a focuscell inlet.

The cell sorting device can comprise a plurality of outlets forcollecting sorted portions of the plurality of cells wherein the sortedportions share one or more biophysical properties. When sorting based onviscosity or viscoelasticity, the cell medium can comprise at least afirst cell portion that is more viscous than at least a second cellportion. The more viscous cell portion can follow a different trajectorythan the less viscous cell portion due to one or more of the pluralityof compression gaps, the ridge spacing, the number of ridges, the ridgeangle, and the flow velocity. The cell sorting device can sort the moreviscous cells from the less viscous cell portion and collect the sortedportions in each outlet of the plurality of outlets. The compressiongap, therefore, can comprise a height smaller than the average diameterof the plurality of cells flowed through the device and the plurality ofcells can go through one or more compressions.

In another exemplary embodiment, the present invention can comprise amicrofluidic device comprising a first wall, a second wall, first andsecond side walls, each extending between the first wall and the secondwall and with the first and second walls, forming a channel, a pluralityof ridges, wherein each ridge of the plurality of ridges extends intothe channel normal from the first wall and defines a compression gapbetween the ridge and the second wall, an inlet for inletting fluid intothe channel, an outlet for outletting fluid from the channel, and anexpansion region disposed downstream from the plurality of ridges,wherein each ridge of the plurality of ridges is diagonally orientedwith respect to a central axis of the microfluidic device, and whereineach pair of adjacent ridges of the plurality of ridges is separated bya ridge spacing having a width of at least 100 microns.

In another exemplary embodiment, the present invention can comprise amicrofluidic device comprising a first wall, a second wall, first andsecond side walls, each extending between the first wall and the secondwall and with the first and second walls, forming a channel, a pluralityof ridges, wherein each ridge of the plurality of ridges extends intothe channel normal from the first wall and defines a compression gapbetween the ridge and the second wall, an inlet for inletting fluid intothe channel, an outlet for outletting fluid from the channel, whereinthe outlet comprises a flow apportionment region, a flow balancingregion, and a collection point, wherein each ridge of the plurality ofridges is diagonally oriented with respect to a central axis of themicrofluidic device, and wherein each pair of adjacent ridges of theplurality of ridges is separated by a ridge spacing having a width of atleast 100 microns.

In another exemplary embodiment, the present invention can comprise amicrofluidic device comprising a first wall and a second wall, formingtherebetween a path for fluid flow, a set of ridges extending into thepath between the first wall and a second wall, an inlet for supplying afluid flow to the path between the first wall and a second wall, anoutlet for removing a fluid flow from the path between the first walland a second wall, and an expansion region disposed upstream from theoutlet and downstream from the set of ridges, and wherein each ridgeextends a ridge distance into the path between the first wall and asecond wall, each ridge distance being less than the distance betweenthe first wall and a second wall, such that each ridge extends from oneof the first and second walls towards the other of the first and secondwalls and forms a compression gap between the ridge and the other of thefirst and second walls toward which the ridge extends, is diagonallyoriented at a ridge angle with respect to a central axis of themicrofluidic device, and is separated from another ridge by a ridgespacing.

In another exemplary embodiment, the present invention comprises a firstplanar wall spaced apart from a second wall by a spaced apart distance,defining therebetween a path for fluid flow, the first wall comprising aplurality of ridges that extend into the spaced apart distance betweenthe first planar wall and the second planar, wherein each ridge of theplurality of ridges extends normal from the first wall and defines acompression gap between the ridge and the planar second wall, an inletfor fluid flow into the spaced apart first and second planar walls, anoutlet for fluid flow out of the spaced apart first and second planarwalls, and an expansion region disposed downstream from the plurality ofridges, wherein each ridge of the plurality of ridges is diagonallyoriented with respect to a central axis of the microfluidic device andeach respective ridge of the plurality of ridges is separated by a ridgespacing having a width of at least 100 microns.

A height of each of the compression gaps can be from about 4 microns toabout 16 microns.

The width of each of the ridge spacings can be from about 100 microns toabout 300 microns.

The width of each of the ridge spacings can be from about 100 microns toabout 200 microns and a ridge angle formed by at least one ridge withrespect to the central axis of the microfluidic device can be about 30degrees.

A ridge angle formed by at least one ridge with respect to the centralaxis of the microfluidic device can be from about 20 to about 75degrees.

The plurality of ridges can comprise from 7 to 21 ridges.

In another exemplary embodiment, the present invention comprises a firstplanar wall spaced apart from a second wall by a spaced apart distance,defining therebetween a path for fluid flow, the first wall comprising aplurality of ridges that extend into the spaced apart distance betweenthe first planar wall and the second planar, wherein each ridge of theplurality of ridges extends normal from the first wall and defines acompression gap between the ridge and the planar second wall, an inletfor fluid flow into the spaced apart first and second planar walls, anoutlet for fluid flow out of the spaced apart first and second planarwalls, and wherein the outlet comprises a flow apportionment region, aflow balancing region, and a collection point, and wherein each ridge ofthe plurality of ridges is diagonally oriented with respect to a centralaxis of the microfluidic device and each respective ridge of theplurality of ridges is separated by a ridge spacing having a width of atleast 100 microns.

The flow balancing region can comprise a serpentine channel.

In another exemplary embodiment, the present invention comprises a firstplanar wall spaced apart from a second planar wall by a spaced apartdistance, defining therebetween a path for fluid flow, the first andsecond planar walls extending a separation length along the path forfluid flow, an inlet for fluid flow into the spaced apart first andsecond planar walls, an outlet for fluid flow out of the spaced apartfirst and second planar walls, a set of ridges extending a ridge lengthalong the path for fluid flow that is less than the separation length,each ridge extending a ridge distance into the spaced apart distancebetween the first planar wall and the second planar, each ridge distancebeing less than the spaced apart distance such that each ridge extendsfrom one of the first and second planar walls towards the other of thefirst and second planar walls and forms a compression gap between theridge and the other of the first and second planar walls toward whichthe ridge extends, wherein each ridge is diagonally oriented withrespect to a central axis of the microfluidic device, and each ridge isseparated from another ridge by a ridge spacing, and an expansion regiondisposed upstream from the outlet and downstream from the ridge length.

A height of each of the compression gaps can be from about 4 microns toabout 16 microns.

A ridge angle formed by at least one ridge with respect to the centralaxis of the microfluidic device can be from about 20 to about 75degrees.

An exemplary embodiment of the present disclosure comprises a method forsorting a plurality of cells including providing a cell medium, the cellmedium comprising a plurality of cells to be sorted, passing the cellmedium through a microchannel having a plurality of diagonally orientedridges, and collecting sorted portions of the cell medium at two or morecollection points. The plurality of diagonally oriented ridges candefine a compression gap between a bottom surface of the microchanneland each ridge of the plurality of ridges. Additionally, when the cellmedium passes through the microchannel, at least a portion of theplurality of cells can undergo one or more compressions due to thecompression gap. Each respective ridge of the plurality of diagonallyoriented ridges can also be separated by a diagonally oriented ridgespacing. The method can have some or all of the features discussed abovewith respect to the microfluidic device and the cell sorting device.

The microchannel can comprise at least two trajectories for theplurality of cells at each ridge. In some exemplary embodiments, thetrajectories can be determined by a characteristic of the cell selectedfrom cell size, stiffness, relaxation time, viscosity, or elasticity,and combinations thereof. In some exemplary embodiments, the cell mediumcan comprise at least a first cell portion that is more viscous than atleast a second cell portion and the more viscous cells can be collectedat a first collection point and the less viscous cell portion can becollected at a second collection point. Additionally, the cell mediumcan be provided to the microfluidic device at a flow velocity of about 3to about 1000 mm/s.

In another exemplary embodiment, the present invention can comprise amethod for sorting cells using a microfluidic device comprisingproviding a cell medium to a microfluidic device comprising a firstwall, a second wall, first and second side walls, each extending betweenthe first wall and the second wall, a plurality of ridges, wherein eachridge of the plurality of ridges extends normal from the first walltoward the second wall, and defines a compression gap between the ridgeand the second wall, and an expansion region disposed downstream fromthe plurality of ridges, wherein each ridge of the plurality of ridgesis diagonally oriented with respect to a central axis of themicrofluidic device, passing the cell medium through the microfluidicdevice, and collecting sorted portions of the cell medium downstream theexpansion region, wherein at least a portion of cells undergo one ormore compressions due to the compression gaps.

In another exemplary embodiment, the present invention comprisesproviding a cell medium through an inlet, the cell medium comprisingcells to be sorted, passing the cell medium through a set of diagonallyoriented ridges, and collecting sorted portions of the cell mediumdownstream an expansion region, wherein at least a portion of cellsundergo one or more compressions due to compression gaps.

The cell medium can comprises a first cell portion and a second cellportion, wherein the first cell portion has a higher viscosity than thesecond cell portion, wherein a microfluidic device comprises twooutlets, a first outlet and a second outlet, and wherein the first cellportion is collected downstream the first outlet and the second cellportion is collected downstream the second outlet.

Each respective ridge of the plurality of diagonally oriented ridges canbe separated by a diagonally oriented ridge spacing, and each ridge ofthe set of ridges can extend from the same planar wall.

The widths of each of the diagonally oriented ridge spacings can have awidth from about 100 microns to about 350 microns, and a height of eachcompression gap can be the same.

A ridge angle formed by at least one ridge with respect to a centralflow axis can be from about 20 degrees to about 75 degrees.

The first cell portion can be collected at a first collection point andthe second cell portion can be collected a second collection point, andat least one collection point can be downstream from a flow balancingregion and a flow apportionment region.

A height of each of the compression gaps can be from about 4 microns toabout 16 microns.

The set of ridges can comprise from 7 ridges to 21 ridges.

These and other objects, features and advantages of the presentdisclosure will become more apparent upon reading the followingspecification in conjunction with the accompanying description, claimsand drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying Figures, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIG. 1A is a cross-sectional diagram of a cell sorting device, inaccordance with an exemplary embodiment of the present disclosure.

FIG. 1B a diagram of a cell sorting device and images showing one ormore cell mixtures infused into a microfluidic channel of a cell sortingdevice, in accordance with an exemplary embodiment of the presentdisclosure

FIG. 2A is a schematic showing a three-outlet, microfluidic cell sortingdevice having a plurality of diagonal ridges, in accordance with anexemplary embodiment of the present disclosure.

FIG. 2B shows various computational fluid dynamics simulations used toengineer hydrodynamic balancing near the outlets, in accordance with anexemplary embodiment of the present disclosure.

FIG. 2C shows a schematic describing cell fractionation of a single celltype (K562) based on the spread of cell stiffness, in accordance with anexemplary embodiment of the present disclosure.

FIG. 2D shows a schematic describing cell fractionation of two celltypes (K562 and HL60) based on the spread of cell relaxation, inaccordance with an exemplary embodiment of the present disclosure.

FIGS. 3A-3D compare the cell diameter, Young's modulus, cell deformationenergy, and cell relaxation constant of HL60 and K562 cells, inaccordance with an exemplary embodiment of the present disclosure.

FIGS. 4A-4B compare the cell lateral displacement between ridges and mapthe trajectory of weakly viscous cells versus highly viscous cells, inaccordance with an exemplary embodiment of the present disclosure.

FIGS. 4C-4D compare the cell relaxation of two cell types in amicrofluidic device with a ridge spacing of L=100 μm and L=200 μm, inaccordance with an exemplary embodiment of the present disclosure.

FIGS. 4E-4F illustrate the effects of larger spacing between ridges andreducing channel flow rate on cell relaxation, in accordance with anexemplary embodiment of the present disclosure.

FIG. 5A shows a flow cytometric analysis of cell enrichment for anexemplary embodiment comprising a 30° ridge angle and a ridge spacing of100 μm, in accordance with an exemplary embodiment of the presentdisclosure.

FIG. 5B shows a flow cytometric analysis of cell enrichment for anexemplary embodiment comprising a ridge spacing of 200 μm, in accordancewith an exemplary embodiment of the present disclosure.

FIGS. 6A-6D compare the cell diameter, Young's modulus, cell deformationenergy, and cell relaxation constant for WBC and K562 cell types, inaccordance with exemplary embodiments of the present disclosure.

FIG. 6E shows a flow cytometric analysis of cell enrichment for anexemplary embodiment having a ridge angle of 30 degrees, a ridge spacingof 200 μm, and a ridge count of 30 ridges, in accordance with exemplaryembodiments of the present disclosure

FIGS. 7A-7E compare the cell diameter, Young's modulus, cell deformationenergy, and cell relaxation constant for two different cell types in athree-outlet microfluidic device, in accordance with exemplaryembodiments of the present disclosure.

FIG. 7F shows a flow cytometric analysis of cell fractionation using athree-outlet microfluidic device, in accordance with exemplaryembodiments of the present disclosure.

FIGS. 8A-8E show an AFM comparison of cells sorted based on celldiameter, Young's modulus, cell deformation energy, and cell relaxation,in accordance with exemplary embodiments of the present disclosure

DETAILED DESCRIPTION OF THE DISCLOSURE

Although preferred exemplary embodiments of the disclosure are explainedin detail, it is to be understood that other exemplary embodiments arecontemplated. Accordingly, it is not intended that the disclosure islimited in its scope to the details of construction and arrangement ofcomponents set forth in the following description or illustrated in thedrawings. The disclosure is capable of other exemplary embodiments andof being practiced or carried out in various ways. Also, in describingthe preferred exemplary embodiments, specific terminology will beresorted to for the sake of clarity.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise.

Also, in describing the preferred exemplary embodiments, terminologywill be resorted to for the sake of clarity. It is intended that eachterm contemplates its broadest meaning as understood by those skilled inthe art and includes all technical equivalents which operate in asimilar manner to accomplish a similar purpose.

Ranges can be expressed herein as from “about” or “approximately” oneparticular value and/or to “about” or “approximately” another particularvalue. When such a range is expressed, another exemplary embodimentincludes from the one particular value and/or to the other particularvalue.

Using “comprising” or “including” or like terms means that at least thenamed compound, element, particle, or method step is present in thecomposition or article or method, but does not exclude the presence ofother compounds, materials, particles, method steps, even if the othersuch compounds, material, particles, method steps have the same functionas what is named.

Mention of one or more method steps does not preclude the presence ofadditional method steps or intervening method steps between those stepsexpressly identified. Similarly, it is also to be understood that themention of one or more components in a device or system does notpreclude the presence of additional components or intervening componentsbetween those components expressly identified.

Microfluidic devices for use in cell sorting are disclosed. In someexemplary embodiments, the microfluidic device can sort a plurality ofcells based on a variety of biophysical and biomechanical properties,including size, viscosity, elasticity, and viscoelasticity. For example,in an exemplary embodiment for cell sorting based on viscoelasticity,the microfluidic device can sort or separate cells into highly viscousand weakly viscous portions. In an exemplary embodiment, the disclosedmicrofluidic devices can comprise a plurality of ridges that arediagonally oriented. Additionally, the disclosed microfluidic devicescan comprise a plurality of compression gaps for constricting the one ormore cells and influencing the trajectory of cells as they progressthrough the microfluidic device. Depending on one or more biophysical orbiomechanical properties, cells can progress through the microfluidicdevice following a unique trajectory. In some exemplary embodiments, themicrofluidic device can comprise an increased number of outlets that canseparate cells into groups having a higher purity.

Various microfluidic devices are described. The described microfluidicdevices can comprise a microchannel defined by a first planar wall and asecond planar wall. The microchannel may comprise a plurality of ridgesprotruding outwardly from a planar wall. The ridge can protrudeperpendicularly (i.e. normal) from one or both of the planar walls, butdoes not necessarily need to. The plurality of ridges can protrude fromone of the first planar wall or the second planar wall. For instance,each of the plurality of ridges can protrude outwardly from the firstplanar wall and towards a second planar wall. In some exemplaryembodiments, the plurality of ridges can protrude outwardly from boththe first planar wall and the second planar wall. For example, each ofthe plurality of ridges can protrude outwardly from the first planarwall towards a second plurality of ridges protruding outwardly from thesecond planar wall.

The microfluidic device may comprise a plurality of diagonally orientedridges. For instance, the one or more ridges may be diagonally orientedwith respect to a central axis of the microfluidic device. The centralaxis may comprise an axis extending parallel to the first planar walland the second planar wall. The plurality of diagonally oriented ridgescan extend parallel to each subsequent ridge of the plurality of ridges.The plurality of diagonally oriented ridges may be straight, but neednot be. Additionally, the plurality of diagonally oriented ridges can beany shape, including but not limited to rectangular, cylindrical,trapezoidal, or triangular.

The microfluidic device can be defined by one or more geometricparameters of the microfluidic device that can be changed as desired.For instance, the plurality of ridges can define a compression gap, aridge spacing, a ridge angle, the number of ridges, a channel length,and a ridge thickness. As used herein, the dimensions of the respectiveelements each reflect an average measurement of the element in thedevice.

Disclosed are a plurality of ridges that may define a compression gapbetween a ridge and a surface of an opposing planar wall. For instance,in an exemplary embodiment wherein the plurality of ridges protrudesfrom the first planar wall, the plurality of ridges may define acompression gap between a ridge and a surface across from the ridge onthe second planar wall. As used herein, a surface may include theclosest or nearest portion of the opposing wall, for example where thewall does not otherwise have corresponding ridges or protrusions. Insome exemplary embodiments the second planar wall can comprise aplurality of ridges, and the opposing surface can be, for example, anopposing ridge. The compression gap can therefore be defined as thespace formed between a ridge and a surface of the second wall, or thespace between opposing ridges on opposing walls. Typically, the opposingridges will be aligned with each other as well. The size of thecompression gap can be increased or decreased as desired, based ondevice design. In some exemplary embodiments, the height of thecompression gap may be from about 4 to about 16 microns, from about 5 toabout 15 microns, from about 5 to about 14, from about 6 to about 14,from about 6 to about 12, or from about 6 to about 11 microns.

The plurality of ridges may be separated by a ridge spacing. The ridgespacing can include the channel or gap formed between respective ridges.The ridge spacing may be increased or decreased as desired, based ondevice design. In some exemplary embodiments, the ridge spacing may befrom about 50 to about 1000 microns, from about 50 to about 750 microns,from about 50 to about 500 microns, from about 50 to about 400 microns,from about 50 to about 350 microns, from about 100 to about 300 microns,from about 100 to about 750 microns, from about 100 to about 500microns, from about 100 to about 400 microns, from about 100 to about300 microns, about 100 to about 250 microns, or from about 125 to about250 microns. The ridge spacing can be at least about 50 microns, atleast about 100 microns, at least about 125 microns, at least about 150microns, at least about 250 microns, or at least about 300 microns. Theridge spacing can be up to about 5 microns, up to about 3 microns, up toabout 2 microns, up to about 1 microns, up to about 750 microns, or upto about 500 microns.

The diagonal orientation of the plurality of ridges with respect to thecentral axis can be defined by a ridge angle. The ridge angle (a) andthe central axis, i.e. the flow direction, can be seen for example inFIG. 1B. The ridge angle may be increased or decreased, based on devicedesign. For instance, in some exemplary embodiments, the ridge angle canbe from about 20 to about 75 degrees, at least about 30 degrees, atleast about 45 degrees, or at least about 60 degrees. The ridge angle ofeach respective ridge may also be the same or different along a lengthof the microfluidic device. In instances where a ridge is not linear,the angle can be measured based on a line that is a linear fit to thenon-linear ridge.

The number of ridges in the microfluidic channel can be increased ordecreased as desired. In some exemplary embodiments, the microfluidicdevice can comprise 5 to 100 ridges. In some exemplary embodiments, themicrofluidic device can comprise at least 3 ridges, at least 4 ridges,at least 5 ridges, at least 6 ridges, at least 7 ridges, at least 8ridges, at least 9 ridges, or at least 10 ridges. In some exemplaryembodiments, the microfluidic device can comprise up to 100 ridges, upto 75 ridges, up to 50 ridges, or up to 40 ridges. In some exemplaryembodiments, the microfluidic device can include 5 to 50 ridges, 7 to 40ridges, or 7 to 21 ridges. In some exemplary embodiments, themicrofluidic device can comprise about 14 ridges.

The plurality of ridges can be described by a ridge thickness. The ridgethickness can be defined as the linear measurement of the ridge in thedirection of primary flow. The ridge thickness can be increased ordecreased as desired, based on device design. In some exemplaryembodiments, the ridge thickness can be from about 7 to about 30microns, from about 7 to about 20 microns, from about 7 to about 18microns, from about 7 to about 16 microns, from about 7 to about 11microns, from about 7 to about 9 microns, from about 20 to about 30microns, from about 22, to about 28 microns, from about 24 to about 28microns, from about 18 to about 21 microns, from about 16 to about 22microns, or from about 8 to about 11 microns. In some exemplaryembodiments the ridge thickness can be at least about 9 microns, atleast about 11 microns, and at least about 16 microns.

The microfluidic device can have one or more inlets. The one or moreinlets may be located on a first side wall of the microfluidic device.In some exemplary embodiments, the microfluidic device can have a cellinlet and a sheath flow inlet. In some exemplary embodiments, the cellinlet can be located between a first sheath flow inlet and a secondsheath flow inlet, or can be surrounded by a first sheath flow inlet. Insome exemplary embodiments, the cell inlet can be downstream from one ormore sheath flow inlets, or can be aligned with one or more sheath flowinlets.

The microfluidic device may comprise two or more outlets. In someexemplary embodiments, the microfluidic device can comprise at least twooutlets, at least three outlets, at least four outlets or at least fiveoutlets. The number of outlets can be two, three, four or five.

The described microfluidic devices can be constructed in a variety ofways. In one exemplary non-limiting exemplary embodiment, themicrofluidic devices can be made using a replica molding ofpolydimethylsiloxane (PDMS) on a permanent mold. The mold can be createdby two-step photolithography patterning of a photoresist on a4-inch-diameter silicon wafer. After the removal of PDMS from the mold,inlet and outlet holes can be punched in the side walls of the PDMS, andthe PDMS can be subsequently bonded to a glass substrate to form themicrofluidic channel.

Exemplary embodiments of the microfluidic device may be used for sortinga plurality of cells (i.e. a cell sorting device) based on one or morebiophysical or biomechanical properties, including but not limited to,size, elasticity, viscosity, and/or viscoelasticity. Cell sorting devicemay comprise some or all of the features discussed above. An exemplarymicrofluidic device for cell sorting 100 is illustrated in FIGS. 1A-1B.The cell sorting device 100 can comprise a top planar wall 110 and abottom planar wall 120. The top planar wall 110 can comprise a pluralityof ridges 130 protruding outwardly from the top planar wall 110. Thecell sorting device 100 can comprise one or more inlets 140 provided forflowing one or more of a cell medium comprising a plurality of cells 180and a sheath flow fluid into the cell sorting device 100. The cellsorting device 100 can comprise a plurality of outlets 150 forcollecting sorted portions of the plurality of cells 180 wherein thesorted portions may share one or more biophysical properties. Top planarwall 110 and bottom planar wall 120 are shown as being made of PDMS andglass, respectively, but are not so limited and can be made of anymaterial known to one of ordinary skill.

The cell sorting device can comprise a plurality of ridges 130 whereinthe ridges are diagonally oriented with respect to a central flow axis,as illustrated at FIG. 1B. The central flow axis can be locatedproximate a central portion of the cell sorting device and comprise anaxis running parallel to a primary flow through the cell sorting device.Each ridge of the plurality of ridges 130 can have a ridge thicknessthat is at least about the average diameter of the cells flowed throughthe cell sorting device, and as discussed previous. Each ridge of theplurality of ridges 130 can define a compression gap 170, as discussedabove. The compression gap 170 can be formed between a ridge 130 and asurface of the bottom planar wall 120. As will be understood, theplurality of cells that are flowed through the cell sorting device cango through a state of compression and a state of relaxation due to thecompression gap 170, and as illustrated at FIG. 1A.

Each compression gap 170 can be sized smaller than the average diameterof a cell, or in other words, as a function of the size of the cell. Forexample, in an exemplary embodiment where the cell medium flowed throughthe cell sorting device comprises T-cells having an average diameter of9 microns, the compression gap can have a height between 5 and 7microns. For example, in an exemplary embodiment where the cell mediumflowed through the cell sorting device comprises stem cells having anaverage diameter of 16 microns, the compression gap can have a heightbetween 8 and 10 microns. For example, in an exemplary embodiment wherethe cell medium flowed through the cell sorting device comprises B-cellshaving an average diameter of 11 microns, the compression gap can have aheight between 6 and 8 microns. In some exemplary embodiments, thecompression gap can have a diameter that is between about 30% to about85% of the diameter of the cells flowed through the cell sorting device,about 40% to about 80% of the diameter of the cells flowed through thecell sorting device, or about 50% to about 75% of the diameter of thecells flowed through the cell sorting device.

The plurality of ridges may be separated by a ridge spacing 160. Theridge spacing 160 can comprise the width of a space or channel formedbetween a first ridge of the plurality of ridges and a second ridge ofthe plurality of ridges. In some exemplary embodiments, the ridgespacing 160 may be from 50 to about 1000 microns, from about 50 to about750 microns, from about 50 to about 500 microns, from about 50 to about400 microns, from about 50 to about 350 microns, from about 100 to about300 microns, from about 100 to about 750 microns, from about 100 toabout 500 microns, from about 100 to about 400 microns, from about 100to about 300 microns, about 100 to about 250 microns, or from about 125to about 250 microns. The ridge spacing can be at least about 50microns, at least about 100 microns, at least about 125 microns, atleast about 150 microns, at least about 250 microns, or at least about300 microns. The ridge spacing can be up to about 5 microns, up to about3 microns, up to about 2 microns, up to about 1 microns, up to about 750microns, or up to about 500 microns, about 50 to about 350 microns, fromabout 100 to about 300 microns, from about 100 to about 250 microns,from about 125 to about 250 microns, or at least 300 microns. Adjustingthe ridge spacing 160 can permit cell sorting by different cellularproperties. In an exemplary embodiment for cell sorting based onviscoelasticity, increasing the spacing between the ridges can beadvantageous because it allows for increased relaxation time for lessviscous cells so they can be sorted from highly viscous cells.

The plurality of ridges may comprise a ridge angle (a), as illustratedat FIG. 1B. The plurality of ridges 130 can be inclined at an angle tocreate hydrodynamic circulations underneath the ridge and can bedesigned to compress and translate cells normal to the flow direction.The ridge angle can also affect the trajectories of cells. The ridgeangle may vary depending on one or more parameters including, but notlimited to, the types of cells flowed through the cell sorting device,the ridge spacing, and the flow velocity of the medium flowed throughthe cell sorting device. As such, adjusting the ridge angle mayfacilitate migration of cells along the ridges, particularly when thecells are less viscous. For instance, increasing the ridge angle maypermit a longer relaxation time for less viscous cells before the lessviscous cells are subjected to a subsequent compression gap. In otherwords, the ridge angle may influence the trajectory of a cell and inturn the sorting capabilities of the cell sorting device.

The cell medium can be flowed into the cell sorting device at a flowvelocity. The flow velocity can be increased or decreased as desired,based on device design. As used herein, the flow velocity can describethe velocity of the cell medium at an inlet or at an outlet. The flowvelocity can be from about 3 to about 1000 mm/s, from about 3 to about500 mm/s, from about 3 to about 250 mm/s, from about 3 to about 100mm/s, from about 3 to about 50 mm/s, from about 3 to about 25 mm/s. Theflow velocity can be at least about 3 mm/s, at least about 20 mm/s, atleast about 50 mm/s, at least about 100 mm/s, or at least about 500mm/s. The flow velocity can be about 3 mm/s, about 20 mm/s, about 500mm/s, or about 1000 mm/s. The flow velocity can also be adjusted as afunction of the length of the channel, and/or the size of the ridgespacing, based on device design. For instance, increasing the length ofthe channel can allow for a greater flow velocity. Increasing thevelocity in similarly sized devices can result in increased pressurewithin the device. By increasing the length of the device, the increasedpressure can be accounted for while permitting higher flow velocity.Increasing the size of the ridge spacing can permit for greater positivelateral displacement in cells having certain biophysical properties(such as increased stiffness or viscosity). For instance, increasing theridge spacing can permit increasing the flow velocity as the greaterspace allows the cells a longer distance over which to travel and besubjected to secondary flow in the ridge channels. As such, increasedridge spacing can permit an increased relaxation time and positivelateral displacement for certain cells despite greater flow velocity.

In some exemplary embodiments, and as illustrated at FIG. 1B, the cellsorting device can comprise one or more sheath flow inlets (or cellfocusing inlets) for flowing a sheath fluid into the cell sortingdevice. A sheath fluid can allow for hydrodynamic focusing of the cellmedium. The one or more sheath flow inlets can be located proximate thecell flow inlet, or upstream of the cell flow inlet. Focusing the cellsin the inlet can comprise providing a sheath fluid to the sheath flowinlet until the sheath fluid reaches laminar flow and then subsequentlyintroducing the cell medium cell medium through the cell inlet. Thecells can be introduced into the cell inlet by injection, for example bysyringe pumps.

When a cell medium is flowed through the microfluidic device, cells,depending on one or more biophysical or biomechanical properties, mayfollow unique trajectories. The trajectories can be determined by thebiophysical or biomechanical properties or characteristic of the cellselected from cell size, stiffness, relaxation time, viscosity, orelasticity, and combinations thereof. For instance, as illustrated atFIG. 1B, after flowing through a compression gap, less viscous cells mayhave a longer relaxation time before flowing beneath a subsequentcompression gap. As such, less viscous cells may be more susceptible tosecondary flow within the channels formed between each respective ridge.For instance, after a less viscous cell is subjected to compression bythe compression gap, the less viscous cell may follow secondary flowwithin the ridge channels and therefore show greater lateraldisplacement down the ridge channels compared to more viscous cells.Therefore, the less viscous cells may follow a unique trajectory fromthe more viscous cells.

The cell sorting device can comprise a plurality of outlets forcollecting sorted portions of the plurality of cells wherein the sortedportions share one or more biophysical or biomechanical properties. Forinstance, in an exemplary embodiment sorting cells based onviscoelasticity, the cell sorting device can comprise two outletswherein one outlet collects highly viscous cells and the second outletcollects less viscous cells. As discussed above, cells having differentbiophysical or biomechanical properties may follow unique trajectories,e.g. cells may travel through the device towards a particular outletbased on one or more biophysical or biomechanical properties. As will beunderstood, increasing the outlets can result in more focused sortingwith increased purity.

The disclosed microfluidic devices, as described above, can also be usedfor high purity cell sorting based on a variety of biomechanicalproperties. For instance, the microfluidic device can sort a pluralityof cells based on one or more of cell stiffness (i.e. cell elasticity),cell viscosity, and cell size. In exemplary embodiments for cell sortingbased on stiffness, viscosity, and/or cell size, the number of outletscan be increased to provide for greater purity in cell sorting. Multipleoutlets can be advantageous in some instances as these outlets canprovide for finer gradation of cells based on cell biomechanicalproperties compared to the binary outputs.

For example, FIG. 2A illustrates a non-limiting example of amicrofluidic device 200 can comprising three outlets (230 a, 230 b, 230c). The microfluidic device 200 can comprise, for example, a top outlet230 a, a central outlet 230 b, and a bottom outlet 230 c. The centraloutlet 230 b can be configured to collect cells that have moderatebiomechanical properties. For instance, in the case of separation of twocell types, the central outlet 230 b can collect those cells that haveoverlapping or moderate biomechanical properties, as illustrated at FIG.2C. As a result, the cells collected at the top 230 a and bottom 230 boutlets can have pronouncedly different biomechanical properties, asillustrated at FIG. 2D.

The disclosed microfluidic devices may comprise an expansion region anda plurality of hydrodynamically balanced outlets. FIG. 2A illustrates anexemplary and non-limiting three-outlet microfluidic device 200comprising an expansion region 240 and three hydrodynamically balancedoutlets (230 a, 230 b, 230 c). The hydrodynamically balanced outlets(230 a, 230 b, 230 c) can each independently comprise a flowapportionment region 260, a flow balancing region 270, and a collectionpoint 280. The expansion region 240 can comprise a ridge-free portion ofthe microfluidic channel 250 comprising the plurality of ridges. Theexpansion region 240 can be in fluid communication with the flowapportionment regions 260 of the outlets (230 a, 230 b, 230 c). Theexpansion region 240 and flow apportionment regions 260 can have anadded benefit of evenly dividing channel flow amongst the outlets (230a, 230 b, 230 c). In some exemplary embodiments, at least one of theoutlets can comprise a flow apportionment region that is larger orsmaller than at least a flow apportionment region of another outlet. Ina non-limiting example, as illustrated at FIG. 2A, the flowapportionment region of the bottom outlet 230 c is larger than the flowapportionment region of the top outlet 230 a and the bottom outlet 230b.

The outlets (230 a, 230 b, 230 c) may also each independently comprise aflow balancing region 270 and a collection point 280. The flow balancingregion 270 can be downstream from and in fluid communication with theflow apportionment region 260. The collection point 280 can bedownstream from and in fluid communication with the flow balancingregion 270. The flow balancing region 270 can be designed so as toincrease the flow resistance in the outlet and prevent flow biasing fromuneven flow apportionment regions and external perturbations. Theoptimal architecture of the expansion region 240, flow apportionmentregions 260, and flow balancing regions 270 can be determined usingcomputational fluid dynamics to design a balanced channel flow egressacross all outlets, as illustrated at FIG. 2B. In a non-limitingexample, and as illustrated at FIG. 2A, the flow balancing regions 270can comprise a substantially serpentine architecture. As will beunderstood, the flow balancing region can comprise a variety of shapes,architectures, and lengths depending on the device design.

In view of the disclosed device described above, the disclosure can alsoinclude methods for sorting a plurality of cells using a microfluidicdevice as described above. A cell medium may be provided to amicrofluidic device, as described above, and sorted using themicrofluidic device. The method can include sorting a plurality of cellsusing the microfluidic device, the method including the steps ofproviding a cell medium, the cell medium comprising the plurality of thecells to be sorted, passing the cell medium through a microchannelhaving a plurality of diagonally oriented ridges, and collecting sortedportions of the cell medium at two or more collection points. Asdiscussed above, the plurality of diagonally oriented ridges can definea compression gap between a bottom surface of the microchannel and eachridge of the plurality of ridges. When the cell medium passes throughthe microchannel, at least a portion of the plurality of cells undergoone or more compressions at the compression gaps.

The method can include collecting the cell medium at two collectionpoints, three collection points, at least three collection points, ormore than three collection points, as further disclosed above. Themethod includes at least two different trajectories for the plurality ofcells at each ridge. The cells can pass along the ridge in the spacebetween two ridges, or the cells can pass under the ridge by undergoingcompression at the compression gap. A certain portion of the cells canpass along each of these trajectories, as discussed above andexemplified in the examples below. The trajectories can be determined bycertain cellular characteristics. Those characteristics can include cellsize, stiffness, relaxation time, viscosity, or elasticity, andcombinations thereof. In particular, the cellular characteristic caninclude viscosity and/or elasticity, or viscosity. In an exemplaryembodiment, the cell medium can include at least a first cell portionthat is more viscous than at least a second cell portion. The moreviscous cells can be collected at a first collection point and the lessviscous cell portion can be collected at a second collection point.

In some exemplary embodiments, the cells undergo a compression in themethod of about 25 to 85% of the average diameter of the cells, about30% to about 85% of the diameter of the cells, about 40% to about 80% ofthe diameter of the cells, or about 50% to about 75% of the diameter ofthe cells flowed through the cell sorting device.

Other aspects of the method can be reflected in the disclosure set forthabove. The cell medium can be provided to the microfluidic device at aflow velocity as disclosed above. The respective ridges of the pluralityof diagonally oriented ridges can be separated by a diagonally orientedridge spacing, as defined above. The ridge angle in the device formedwith respect to a central flow axis can be as described above. Thecompression gap can be smaller than the average cell diameter of thecell flowing through the device, and can be at heights as describedabove. The plurality of diagonally oriented ridges can include from atleast 5 to 100 or more ridges, and can be as defined above. And themethod can be operated in a device that includes expansion regions, aflow balancing region and a flow apportionment region, as describedabove. Each of the device elements set forth above and expanded beloware incorporated here in the method as well.

EXAMPLES Example 1-Device For Cell Sorting Based On Viscoelasticity

Designing Microfluidic Channels For Sorting Based On Viscoelasticity

The parameters defining the microfluidic channel can be determined usingcomputational fluid dynamics. Cell dynamic mechanical response tocompression can be divided into two components characterized by astorage modulus (elastic, G′) and a loss modulus (viscous, G″). Sincethe channel ridges can compress all cells to the same size, differencesin cell size result in different amounts of deformation. To account fordifferences in cell size, cells can be characterized by a parameterrelated to cell deformation energy to group the cell elasticity and cellsize into size-adjusted elasticity. In this manner, the cell elasticitycan be normalized with respect to cell size. A time-independent Young'smodulus and cell diameter can be applied to represent elastic modulusand cell size respectively. To determine the elastic deformation energy,the Hertzian contact mechanics model can be applied in which theequivalent elastic modulus E* as set forth in Equation 1 (Eq. 1). Wherev is the Poisson's ratio and E is the Young's modulus.

$\begin{matrix}{\frac{1}{E*} = {\frac{1 - v_{cell}^{2}}{E_{cell}} + \frac{1 - v_{channel}^{2}}{E_{channel}}}} & \left( {{Eq}.\mspace{14mu} 1} \right)\end{matrix}$

Modeling the deformed cell as an elastic sphere, the deformation energy,U, representing size-adjusted elasticity, can be derived by integratingthe compression force, F, over the cell deformation S as set forth inEquation 2 (Eq. 2), where S is the amount of deformation of a compressedcell, estimated as the difference between cell diameter, d, and ridgegap, h. As a result, the deformation energy can incorporate both cellelasticity and cell size.

$\begin{matrix}{U = {{\int{FdS}} = {\int{\frac{4}{3}E*\left( \frac{d}{2} \right)^{0.5}(S)^{1.5}dS}}}} & \left( {{Eq}.\mspace{14mu} 2} \right)\end{matrix}$

A model was created to relate the biophysical properties to celltrajectory in the microfluidic cell sorter. The model describing thecell trajectory in the microfluidic channel depends on three factors:the size-adjusted elasticity (cell deformation energy), the cellviscosity, and the strength of the secondary flow, which is representedby the ratio of non-axial volumetric flow to axial volumetric flow. Thesize-adjusted elasticity and viscosity are biophysical properties of thecell. The strength of the helical secondary flow is controlled by thechannel geometry and flow rate. As a result of these factors, celltrajectory under the ridge mostly depends on the cell biophysicalproperties while cell trajectory in between ridges is controlled by thestrength of the secondary flow.

Since the cells are viscoelastic, their dynamic response aftercompression is time dependent. Experiments show that cell relaxationdepends not only on a short time scale characterizing sequentialcompressions but also on a longer time scales due to mechanical changeswithin cells resulting from repeated compressions. It was assumed thatthe viscoelastic response of cells can vary over the course of repeatedcell compression events in the cell sorting microchannel.

Differences in cell viscosity can be exploited by engineering ridgegeometry and spacing to take advantage of cell relaxation. As a cellinitially confronts the ridge, it resists deformation through positivelateral displacement while squeezing through the gap space. However,after compression, a highly viscous cell remains deformed, while aweakly viscous cell relaxes more fully before entering a subsequentridge. Therefore, we can expect that the trajectory of the highlyviscous cells is primarily dominated by the secondary flow while lessviscous cells are dominated by the deformation energy due to thecompressions. Consequently, if the ridge spacing is expanded to allowthe cell sufficient time to relax and to restore the original undeformedshape, the trajectory of a highly viscous cell will again be similar toa weakly viscous cell. A microfluidic device can be designed using theseprinciples.

Fabrication Of Microfluidic Channels

Microfluidic channels with periodic slanted ridges were fabricated bypermanently bonding molded PDMS (Sylgard 184, Dow Corning) to a glasssubstrate. The microfluidic channel features were formed by replicamolding of PDMS on a reusable SU-8 mold (SU-8 2007, Microchem Corp.)formed using two-step photolithography on a four-inch diameter siliconwafer. PDMS base and a curing agent were mixed at 10:1 ratio and poureddirectly onto the SU-8 mold. After degassing, the PDMS was cured at 60degree Celsius for 6 hours. After curing, the PDMS was removed from theSU-8 mold and the inlet and outlet holes were punched, along with otherfeatures of the microfluidic channels. Prior to bonding, themicrofluidic feature dimensions were measured with 3D laser confocalmicroscopy technique (LEXT Olympus). PDMS was treated with air plasma(PDC-32G Harrick) and bonded to a glass slide to form the microfluidicchannel. After bonding, the channel was incubated at 60 degree Celsiusfor an hour. To prevent non-specific cell adhesion to the microfluidicchannel walls, the microfluidic channel was primed with bovine serumalbumin (Sigma Aldrich) at concentration of 10 mg per mL and incubatedat 4 degree Celsius overnight.

Materials And Flow Experiment Setup

K562 cells (CCL-243) and HL60 cells (CCL-240) were purchased from ATCC.K562 and HL60 cells were cultured and maintained in Iscove's modifiedDulbecco's medium (ATCC) with the addition of 10% fetal bovine serum(FBS). Cells were stored at 37 degree Celsius with 5% CO₂. Cells wereexpanded to 0.5 million cells per mL in a culture flask over two days.Whole blood was withdrawn from healthy donors. White blood cells wereseparated from fresh whole blood using Ficoll-Paque (1.077, GE LifeSciences) through centrifugation. The remaining red blood cells werelysed using red blood cell lysis buffer for human (Alfa Aesar). Theisolated white blood cells were from 1 to 2 million cells per mL ofwhole blood and were resuspended in DPBS. For characterization ofsorting by flow cytometric analysis (Accuri C6, BD), cells were labeledwith lipid stains (Bybrand, Life Technologies) at 5uL reagent per mL ofcell suspension.

Cell mixtures at concentration from 1 to 2 million cells per mL werecontained in a 3 mL syringe and infused into the microfluidic channelusing syringe pumps (PHD 2000, Harvard Apparatus) at controlled flowrates. The cell trajectories were observed by mounting the microfluidicchip on an inverted microscope (Eclipse Ti, Nikon) and recorded byhigh-speed camera (Phantom v7.3, Vision Research) at a frame rate of2000 frames per second. The high-speed videos were analyzed with acustomized algorithm in ImageJ to extract cell trajectories.

Cell stiffness and relaxation rate data were measured using an atomicforce microscope (AFM, MFP-3D, Asylum Research). All cells were measuredwith a rounded cell shape to closely resemble the morphology within themicrofluidic channel. A monolayer of poly-L-lysine (MW 300 k, SigmaAldrich) to serve as anchors to slightly attach cells to the glasssubstrate to improve cell stability during AFM measurement. The cellstiffness was represented by the average young's modulus. Beaded siliconnitride cantilevers (spring constant 37.1 pN per nm) were used to indentthe center of cells at 1.5 μm per second. Sufficient force was appliedto achieve at least 4 μm deformations such that it was in closecomparison with our microfluidic compression. Each cell wascharacterized by three force-indentation curves and fit to a Hertzianmodel to compute the average Young's modulus. The cell viscosity wascharacterized by the relaxation rate constant. After maximum indentationof the cell, the tip was held in place while the compression force wasmonitored for 10 seconds so that cell relaxation can be measured. Cellrelaxation was fit to an exponential function and the relaxation rateconstant of the cell was calculated.

Cell Biophysical Properties Of HL60 And K562 Cells

Two leukemia cell lines, K562 and HL60, were selected to demonstratemicrofluidic sorting of cells based on differences in cell viscosity.These two cell types have differences in cell size (FIG. 3A) and cellelasticity (FIG. 3B), yet exhibit similar size-adjusted elasticity(deformation energy) (FIG. 3C). The cell relaxation rate measured withAFM indicated the K562 cells were less viscous compared to HL60 cells(FIG. 3D).

Effect Of Channel On Lateral Displacement

The lateral cell trajectory within the microfluidic channel can becomposed of two segments: lateral displacement at the ridge and lateraldisplacement between the ridges (FIG. 4A). At the ridge, the lateraldisplacement mainly depends upon the cell biophysical properties sincethe cell body must deform to squeeze through the space between the ridgeand bottom substrate. The lateral displacement at the ridge has at leasttwo components. Depending on the cell stiffness, the resistance todeformation by the leading edge of the ridge may cause call movementeither along the ridge or perpendicular to the ridge. When cellscompletely reside underneath the ridge, they move perpendicular to theridge in response to the flow field regardless of the biophysicalproperties because the compression is complete. As such the cell lateraldisplacement can be characterized by the total displacement at the ridgeas the cells touch the leading edge of the ridge and immediately afterthe cells completely exit the ridge. In between the ridges, the lateraldisplacement depends on the strength of the circulation induced by theridge. The circulation generated by the ridges results in positive celllateral displacement independent of cell viscoelastic properties (FIG.4B).

Channel Parameters

Several geometric parameters of the channel were examined to determinetheir effect including compression gap size (h), ridge angle (α), ridgespacing (L), and number of compressions. The magnitude of the secondaryflow increases with increasing ratio of the ridge height and channelheight. All channels used the same channel height (H=20 μm), thus thesmaller gap size results in stronger secondary flow. The effect ofmagnitude of the secondary flow on cell trajectory is not significantlyfor HL60 and K562 cells as the lateral displacement between ridges didnot differ as evidenced in TABLE 1. As a consequence, the secondary flowis not a major contributing factor to cell separation.

TABLE 1 K562 Cell Lateral Displacement Per Ridge (μm) Channel design 1stridge 10th ridge 28th ridge 68th ridge Between ridge 30° ridge (L = 100μm) 0.89 ± −23     6.04 ± 2.80 −6.08 ± 2.86 −7.95 ± 2.58 7.42 ± 1.36 45°ridge (L = 78 μm) 0.31 ± 0.067 −5.88 ± 2.74 −5.94 ± 2.60 N/A 7.08 ± 1.2930° ridge (L = 200 μm) 0.91 ± 0.34  −3.56 ± 2.31 −5.61 ± 2.41 N/A 10.1 ±0.9  HL60 Cell Lateral Displacement Per Ridge (μm) Channel design 1stridge 10th ridge 28th ridge 68th ridge Between ridge 30° ridge (L = 100μm) −4.03 ± 0.94   −11.1 ± 2.44 −11.4 ± 1.83 −12.1 ± 1.58 7.87 ± 0.6245° ridge (L = 78 μm) −4.08 ± 0.68  −10.82 ± 1.63 −11.2 ± 1.48 N/A 7.52± 0.54 30° ridge (L = 200 μm) −4.11 ± 1.75   −11.2 ± 2.91 −11.6 ± 2.01N/A 11.1 ± 0.51 7.5 μm Particle Lateral Displacement Per Ridge (μm)Channel design Ridge Between ridge 30° ridge (L = 100 μm) −12.0 ± 0.7910.4 ± 1.05 45° ridge (L = 78 μm) −13.3 ± 1.15 12.4 ± 1.82 30° ridge (L= 200 μm) −14.7 ± 1.61 14.1 ± 2.52

Increasing the ridge spacing (L), resulted in increased relaxation ofweakly viscous cells, which could be used to separate cellsviscoelastically. Cell relaxation can be measured by recording theapparent diameter of the compressed cells between first ridge and secondridge in a 9 μm gap channel (h=9 μm, L=100 μm and 200 μm) for bothweakly viscous K562 and highly viscous HL60 cells. In both cases, theless viscous K562 cells relaxed more quickly than HL60 cells, inagreement with the AFM measurements, as shown in FIGS. 4C and 4D. Inaddition, the increased ridge spacing may allow both cell types a longerduration to relax between compressions amplifying the difference inrelaxation between the K562 and HL60 cells (FIG. 4E). The additionalrelaxation resulted from changing the ridge spacing from L=100 μm to 200μm is shown for K562 cells in FIG. 4E. Moreover, the additionalrelaxation increased K562 cell lateral displacement, but the effect wasmore significant than for HL60 cells (FIG. 4E). Therefore, the increasedridge spacing did not result in appreciable cell relaxation of thehighly viscous HL60 cells. As a result, the increased ridge spacingenhanced the divergency of lateral displacement between K562 and HL60cells.

Another parameter that can affect cell trajectory is the ridge angle.When the ridge angle is more aligned with the channel axis (smaller α),elastic cells tend to roll at the leading edge of the ridges. K562 cellsdisplay more positive lateral displacement at the first ridge when theridge is at 30° compared to 45° as evidenced in TABLE 1. Unlike K562cells, the ridge angle did not strongly affect viscous HL60 celltrajectory because HL60 cells remained deformed and had little contactwith the ridges.

For both K562 and HL60 cells, repeated compressions result in negativelateral displacement after 10 ridges as evidenced in TABLE 1. A possiblemechanism for this observed behavior could be that cell relaxation is atime dependent dynamic response to such periodic compression. As suchthe change in cell viscoelastic behavior in the cannel could beattributed to alteration of the cytoskeleton, possibly by disassembly ofstress fiber networks or buckling and disintegration of stress fibers.

The channel flow rate can also be used to affect cell trajectory. In onecase, reducing the flow rate to Q=0.025 mL per minute from Q=0.05 mL permin led to cells having similar relaxation in L=100 μm channel comparedto L=200 μm channel at 0.05 mL per minute (FIG. 4E). While a higher flowrate can increase throughput, viscoelastic separation can be negativelyimpacted by flow rates which result in insufficient cell relaxation.Preferably a flow rate of 0.05 mL per minute may be used to producesufficient differential relaxation and cell sorting of HL60 and K562cells.

As cells undergo initial compression, the cell trajectory is dominatedby size-adjusted elasticity. However, as cell progress through thechannel and rapidly compressed by sequential ridges, cell viscositywhich sets cell relaxation time plays a more important role. When cellrelaxation time is much longer than the time between compressions, thedeformed cells are in disc-shape and their trajectory is primarilyaffected by the secondary flow induced by the diagonal ridges. Thus,cell size, cell stiffness and cell relaxation can be used to designmicrofluidic channels to sort cells primarily based on cell viscousproperties.

Cell enrichment of K562 and HL60

The K562 and HL60 cell lateral displacement and pair-wise separationresults are summarized in TABLE 1 and TABLE 2 respectively.Additionally, flow cytometry was used to verify the improvement in cellseparation and derive a cell enrichment factor. The cell enrichmentfactor normalizes the separated cell populations with the initial cellmixture to obtain enrichment. The HL60 enrichment factor is set forth inEquation 3 (Eq. 3).

$\begin{matrix}{{c.e.f_{HL60}} = \frac{\left( {HL6{0/K}526} \right)_{{HL}\; 60{outlet}}}{\left( {HL6{0/K}526} \right)_{{initial}\mspace{14mu} {mixture}\mspace{14mu} {inlet}}}} & \left( {{Eq}.\mspace{14mu} 3} \right)\end{matrix}$

TABLE 2 Cell Enrichment Factor Channel Design K562 HL60 30° ridge, L =100 μm, 30 ridges 3.26 2.29 45° ridge, L = 78 μm, 30 ridges 2.07 2.8530° ridge, L = 100 μm, 70 ridges 1.15 1.75 30° ridge, L = 200 μm, 30ridges 6.34 4.04

Cell separation of HL60 and K562 mixtures was compared using two channeldesigns. The channel with ridge spacing L=200 μm had a cell enrichmentfactor of 6.34 and 4.04 for K562 and HL60 cells respectively. Comparedto L=100 μm, the enrichment was improved by more than 90% for K562 and75% for HL60. Cell enrichment data for other these and other channeldesigns are summarized in TABLE 2.

Cell enrichment of K562 and healthy leukocytes

Application of this microfluidic approach to other cell lines isenvisioned, and was further demonstrated to enrich K562 leukemia cellfrom healthy white blood cells. The white blood cells (WBC) wereisolated from whole blood of healthy adults and suspended in salinesolution. AFM microscopy was used to measure the white blood cellstiffness and relaxation time constant. Cell diameter of WBC populationis smaller than the K562 cells (FIG. 5A). The WBC have higher Young'smodulus (FIG. 5B). K562 cells have higher size-adjusted elasticity(deformation energy) primarily due to large cell diameter since itscales to the quartic power (FIG. 5C). Similar to HL60, the WBC arehighly viscous cells (FIG. 5D). Coefficients of variation of cell size,stiffness and viscosity distributions are more pronounced, which couldbe a result of WBC population consisting of multiple cell types.

Using a channel design (α=30°, L=200 μm, 30 ridges), flow cytometricanalysis indicated a 5.3-fold K562 enrichment (FIG. 5E). WBC displaylower size-adjusted elasticity and similar viscosity compared to HL60cells. Further testing using alternative channel designs was alsoperformed.

Example 2—Device for Cell Separation and Fractionation

Two types of leukemia cell lines: K562 and HL60 may be sorted using adevice incorporating three outlet channels. The biomechanical propertiesof these two cell types and the results of cell separation using abinary output channel have been characterized. Additionally, the cellsize, Young's modulus, size-adjusted elasticity (deformation energy) andthe relaxation rate constants are given (FIGS. 6A-6D). The average celldiameter for K562 and HL60 cells is 15.5±1.7 μm (n=114) and 12.4±1.2 μm(n=36). AFM measurements of cells' Young's moduli show that HL60(E=0.86±0.22 kPa, n=24) is stiffer than K562 (E=0.34±0.21 kPa, n=114).The average values of Young's modulus and cell diameter are used tocalculate cell deformation energy. K562 and HL60 have similardeformation energy (69.8±26.1, and 67.6±14.5 kPa μm³, respectively) whencompressed with 9-μm channel gap height. AFM measurements of cellrelaxation show HL60 (1.45±0.65 s⁻¹, n=30) is slower in size recoverythan K562 (4.42±3.15 s⁻¹, n=52). t test was used to analyze statisticalsignificance: **p<0.001, ***p<0.0001 and ns no significance. Thesize-adjusted elasticity accounts for cell size and cell stiffness as asingle parameter.

K562 and HL60 cells are primarily sorted based on differences in cellrelaxation due to a higher viscosity of HL60 cells. Cell mixtures arelabeled fluorescently and mixed at 1-2 million cells per mL. The flowrate ranges from 0.0125 to 0.025 mL per min. The cell sorting resultwith the cell fractionation is evaluated by flow cytometry. Flowcytometric analysis of cell enrichment was assessed using a three-outletchannel. At the outlets, the cell enrichment factor for K562 and HL60cells is 45.3 and 15.6, respectively. The purity of enriched K562 cellsand HL60 cells is 98.7 and 87.9%, respectively. The total number ofcells initially is 22,000. The number of cells in the K562 outlet, themiddle outlet and the HL60 outlet is 11,500, 4600, and 2100,respectively, and rounded the nearest hundred. To quantify cell sortingefficiency, the cell enrichment factor (Eq. 3) can be used. The cellenrichment factor accounts cell enrichment by normalizing ratios of cellproportions at outlets to the original cell proportion in the initialmixture.

For the three-outlet device, the cell enrichment factor for K562 andHL60 cells is 45.3 and 15.6, respectively. The fractionation improvesthe cell enrichment factor by an order of magnitude compared to theresults utilizing binary outlets. The additional outlet in the middlecollects those K562 and HL60 cells that have overlapping biomechanicalproperties. Therefore, the three-outlet channel enables a dramaticimprovement in cell sorting purity.

In addition to enrichment of cell mixtures into individual cell types,the three-outlet channel can also be applied to fractionate a singlecell type into distinct biomechanical phenotypes. Since biological cellsare inherently heterogeneous in nature, their biomechanical propertiesmay include large variations due to differences in cytoskeleton ornucleus. For example, K562 cells have average Young's modulus 0.34 kPaand a standard deviation 0.21 kPa. Utilizing the fractionation approach,we are able to obtain subpopulations of K562 cells with differingbiomechanical properties.

K562 cells were fractionated using the three-outlet channel (outlet A,B, and C), and the separated cells were characterized with AFM andoptical microscopy immediately after collection. The biomechanicalproperties of the K562 cells at inlet are characterized. Atomic forcemicroscopy measurements of cell Young's modulus show that sorted K562cells have different average Young's modulus outlet A (E=0.47±0.21 kPa,n=27), outlet B (E=0.33±0.24 kPa, n=53), and outlet C (E=0.25±0.098 kPa,n=38). The average Young's modulus at the inlet is 0.39±0.21 kPa(n=110). Spearman's correlation analysis of the separated cells producesa p value of 0 and an r value of −0.42. Cell sizes are different atthree outlets with the largest average cell diameter in outlet A and thesmallest average cell diameter in outlet C. The cell diameters in outletA, B, C and inlet are 16.28±1.4 μm (n=27), 15.42±1.78 μm (n=53),14.98±1.56 μm (n=38) and 15.2±1.2 μm (n=110), respectively. Spearman'scorrelation analysis of the separated cells produces a p value of 0.001and a r value of −0.29. These data show that K562 cells can besuccessfully separated into three populations with differentbiomechanical properties, predominantly stiffness and size, but notsignificantly by cell relaxation. The size-adjusted elasticity(deformation energy) that combines the effects of cell size andstiffness is also significantly different between the cells collected atthree outlets.

EXEMPLARY EMBODIMENTS

Additionally, or alternately, the disclosure can include one or more ofthe following exemplary embodiments.

Exemplary embodiment 1. A microfluidic device comprising one or moreinlets, a first wall and a second wall, the walls being substantiallyplanar to each other and the first wall having a plurality of ridgeswherein each ridge of the plurality of ridges protrudes normal to thefirst wall and defines a compression gap between the ridge and a surfaceof the second wall, and two or more outlets, wherein each ridge of theplurality of ridges is diagonally oriented with respect to a centralaxis of the microfluidic device and each respective ridge of theplurality of ridges is separated by a ridge spacing.

Exemplary embodiment 2. A cell sorting device for sorting a plurality ofcells based on one or more biophysical cellular properties includingsize, elasticity, viscosity, and/or viscoelasticity, the cell sortingdevice comprising an inlet for flowing a cell medium comprising theplurality of cells into the device at a flow velocity, a top planar walland a bottom planar wall wherein the top planar wall comprises aplurality of ridges protruding normal to the top planar wall anddefining a compression gap between a surface of the bottom planar walland each ridge of the plurality of ridges, and a plurality of outletsfor collecting sorted portions of the plurality of cells wherein thesorted portions share one or more biophysical properties, wherein eachridge of the plurality of ridges is oriented diagonally with respect toa central flow axis and each respective ridge of the plurality of ridgesis separated by a diagonally oriented ridge spacing.

Exemplary embodiment 3. A method for sorting a plurality of cells usinga microfluidic device, the method including the steps of providing acell medium, the cell medium comprising the plurality of the cells to besorted, passing the cell medium through a microchannel having aplurality of diagonally oriented ridges, and collecting sorted portionsof the cell medium at two or more collection points. The plurality ofdiagonally oriented ridges define a compression gap between a bottomsurface of the microchannel and each ridge of the plurality of ridges.When the cell medium passes through the microchannel, at least a portionof the plurality of cells can undergo one or more compressions due tothe compression gap.

Exemplary embodiment 4. The methods or devices of one of the previousexemplary embodiments, wherein the height of the compression gap isabout 4 to about 16 microns, is about 5 to about 14 microns, or is about6 to about 11 microns.

Exemplary embodiment 5. The methods or devices of one of the previousexemplary embodiments wherein the width of the ridge spacing is about 50to about 350 microns, about 100 to about 300 microns, about 100 to about200 microns, or about 100 microns or less.

Exemplary embodiment 6. The methods or devices of one of the previousexemplary embodiments wherein the width of the ridge spacing is about100 to about 200 microns and a ridge angle formed by at least one ridgewith respect to the central axis of the microfluidic device is about 30degrees, or wherein the width of the ridge spacing is about 100 micronsor less and a ridge angle formed by at least one ridge with respect tothe central axis of the microfluidic device is about 45 degrees.

Exemplary embodiment 7. The methods or devices of one of the previousexemplary embodiments wherein a ridge angle formed by at least one ridgewith respect to the central axis of the microfluidic device is about 20to about 75 degrees, or wherein a ridge angle formed by at least oneridge with respect to the central axis of the microfluidic device isabout 30 to about 60 degrees.

Exemplary embodiment 8. The methods or devices of one of the previousexemplary embodiments wherein the plurality of ridges comprises at least7 ridges, 7 to 21 ridges, or 14 ridges.

Exemplary embodiment 9. The methods or devices of one of the previousexemplary embodiments comprising two outlets, at least two outlets,three outlets, at least three outlets, five outlets, or at least fiveoutlets.

Exemplary embodiment 10. The methods or devices of one of the previousexemplary embodiments further comprising an expansion region downstreamfrom the plurality of ridges.

Exemplary embodiment 11. The methods or devices of one of the previousexemplary embodiments wherein at least one outlet comprises a flowapportionment region, a flow balancing region, and a collection point.

Exemplary embodiment 12. The methods or devices of one of the previousexemplary embodiments wherein at least one outlet comprises a flowapportionment region that is a different size than at least a secondflow apportionment region.

Exemplary embodiment 13. The methods or devices of one of the previousexemplary embodiments wherein at least one collection point isdownstream from a flow balancing region and a flow apportionment region.

Exemplary embodiment 14. The methods or devices of one of the previousexemplary embodiments wherein at least one outlet comprises a flowbalancing region that is a serpentine channel.

Exemplary embodiment 15. The methods or devices of one of the previousexemplary embodiments wherein the width of the ridge spacing is about200 microns, a ridge angle formed by at least one ridge with respect tothe central axis of the microfluidic device is about 30 degrees, and theplurality of ridges comprises 30 ridges.

Exemplary embodiment 16. The methods or devices of one of the previousexemplary embodiments wherein the flow velocity is about 3 to about 1000mm/s.

Exemplary embodiment 17. The methods or devices of one of the previousexemplary embodiments wherein the width of the ridge spacing is about100 to about 300 microns and the predetermined flow velocity is about 3to about 1000 mm/s.

Exemplary embodiment 18. The methods or devices of one of the previousexemplary embodiments wherein the width of the ridge spacing is about100 to about 300 microns, the flow velocity is about 3 to about 1000mm/s, and a ridge angle formed by at least one ridge with respect to thecentral flow axis is about 20 to about 75 degrees.

Exemplary embodiment 19. The methods or devices of one of the previousexemplary embodiments further comprising one or more sheath flow inletsfor flowing a sheath fluid into the cell sorting device.

Exemplary embodiment 20. The methods or devices of one of the previousexemplary embodiments wherein the height of the compression gap issmaller than an average cell diameter.

Exemplary embodiment 21. The methods or devices of one of the previousexemplary embodiments wherein the cell medium comprises at least a firstcell portion that is more viscous than at least a second cell portion,wherein the more viscous cell portion follows a different trajectoryfrom the less viscous cell portion.

Exemplary embodiment 22. The methods or devices of one of the previousexemplary embodiments the cell medium comprises at least a first cellportion that is more viscous than at least a second cell portion,wherein the more viscous cell portion follows a different trajectoryfrom the less viscous cell portion, and the more viscous cell portion iscollected at a first outlet and the less viscous cell portion iscollected at a second outlet.

Exemplary embodiment 23. The methods or devices of one of the previousexemplary embodiments wherein at least one outlet comprises a collectionpoint and a more viscous cell portion is collected at a first collectionpoint and a less viscous cell portion is collected at a secondcollection point.

Exemplary embodiment 24. The methods or devices of one of the previousexemplary embodiments wherein collecting sorted portions of the cellmedium occurs at two collection points, or wherein collecting sortedportions of the cell medium occurs at three collection points.

Exemplary embodiment 25. The methods or devices of one of the previousexemplary embodiments wherein the device or microchannel comprises atleast two trajectories for the plurality of cells at each ridge.

Exemplary embodiment 26. The methods or devices of one of the previousexemplary embodiments wherein at least a portion of the plurality ofcells undergoes one or more compressions due to the compression gap.

Exemplary embodiment 27. The methods or devices of one of the previousexemplary embodiments wherein a cell trajectory through the microchannelor device is determined by a characteristic of the cell selected fromcell size, stiffness, relaxation time, viscosity, or elasticity, andcombinations thereof.

Exemplary embodiment 28. The methods or devices of one of the previousexemplary embodiments wherein the cells undergo a compression of about25 to about 75% of the average diameter of the cells.

It is to be understood that the exemplary embodiments and claimsdisclosed herein are not limited in their application to the details ofconstruction and arrangement of the components set forth in thedescription and illustrated in the drawings. Rather, the description andthe drawings provide examples of the exemplary embodiments envisioned.The exemplary embodiments and claims disclosed herein are furthercapable of other exemplary embodiments and of being practiced andcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein are for the purposes ofdescription and should not be regarded as limiting the claims.

Accordingly, those skilled in the art will appreciate that theconception upon which the application and claims are based can bereadily utilized as a basis for the design of other structures, methods,and systems for carrying out the several purposes of the exemplaryembodiments and claims presented in this application. It is important,therefore, that the claims be regarded as including such equivalentconstructions.

1. A method for sorting cells based on viscoelasticity of the cells,using a microfluidic device, the method comprising: providing a cellmedium to the microfluidic device, the microfluidic device comprisingridges, a wall, and an expansion region; wherein the ridges extendtoward the wall and defining compression gaps between the ridges and thewall; and wherein the expansion region is disposed downstream from theridges; passing the cell medium through a portion of the microfluidicdevice comprising the compression gaps; wherein the ridges causeseparation of the cells into sorted portions based on theviscoelasticity of the cells defining ability of the cells to passthrough the compression gaps; and collecting the sorted portions of thecell medium downstream the expansion region.
 2. The method of claim 1,wherein the ridges further cause the separation of the cells into thesorted portions based on one or both of viscosity of the cells and sizeof the cells.
 3. The method of claim 1, wherein the microfluidic devicefurther comprises a first outlet and a second outlet; wherein collectingthe sorted portions of the cells comprises collecting a first one of thesorted portions into the first outlet and collecting a second one of thesorted portions into the second outlet; and wherein the viscoelasticityof the cells in the first one of the sorted portions is different fromthe viscoelasticity of the cells in the second one of the sortedportions.
 4. The method of claim 1 further comprising flowing a sheathfluid into the microfluidic device such that the sheath fluidhydrodynamically focuses the cell medium prior to passing the cellmedium through the portion of the microfluidic device comprising thecompression gaps.
 5. The method of claim 1, wherein the ridges arediagonally-oriented relative to a central axis of the microfluidicdevice, corresponding to a general flow direction of the cell mediumthrough the microfluidic device.
 6. The method of claim 1, wherein theridges extend parallel to each other.
 7. The method of claim 1, whereineach of the ridges is straight.
 8. The method of claim 1, wherein across-sectional profile of each of the ridges is one of rectangular,cylindrical, trapezoidal, or triangular.
 9. The method of claim 1,wherein a thickness of each of the ridges is from about 7 to about 30microns.
 10. The method of claim 1, wherein the microfluidic devicecomprises polydimethylsiloxane (PDMS) and a glass substrate.
 11. Themethod of claim 3, wherein the first outlet and the second outlet arehydrodynamically balanced.
 12. The method of claim 3, wherein the firstoutlet comprises a first flow apportionment region; and wherein thesecond outlet comprises a second flow apportionment region, larger thanthe first flow apportionment region.
 13. The method of claim 3, whereinat least one of the first outlet or the second outlet comprises a flowbalancing region, having a serpentine architecture.
 14. The method ofclaim 4, wherein flowing the sheath fluid into the microfluidic deviceis initiated prior to providing the cell medium to the microfluidicdevice; and wherein the cell medium is provided to the microfluidicdevice after the sheath fluid establishes a laminar flow.
 15. The methodof claim 4, wherein the microfluidic device further comprises a cellflow inlet and one or more sheath flow inlets; wherein providing thecell medium to the microfluidic device comprises flowing the cell mediumthrough the cell flow inlet; and wherein flowing the sheath fluid intothe microfluidic device comprising flowing the sheath fluid through theone or more sheath flow inlet.
 16. The method of claim 15, wherein theone or more sheath flow inlets are positioned upstream from the cellflow inlet.
 17. A method for processing cells using a microfluidicdevice, the method comprising: providing a cell medium to themicrofluidic device, the microfluidic device comprising ridges, a wall,and an expansion region; wherein the ridges extend toward the wall anddefining compression gaps between the ridges and the wall; wherein eachadjacent pair of the ridges is separated by one of ridge spacings; andwherein the expansion region is disposed downstream from the ridges;passing the cell medium through a portion of the microfluidic devicecomprising the compression gaps; wherein the ridges cause compression ofthe cells based on the viscoelasticity of the cells, wherein theviscoelasticity corresponds to ability of the cells to pass through thecompression gaps; and wherein the ridge spacings allow relaxation of thecells based on the viscoelasticity of the cells; and collecting thecells downstream the expansion region.
 18. The method of claim 17,wherein a length of each of the ridge spacings is between 50 micrometersand 1000 micrometers.
 19. The method of claim 17, wherein a length ofeach of the ridge spacings is selected based on variations of theviscoelasticity of the cells.
 20. A microfluidic device for processingcells, the microfluidic device comprising: ridges; ridge spacings,wherein each adjacent pair of the ridges is separated by one of theridge spacings; a wall, wherein the ridges extend toward the wall anddefining compression gaps between the ridges and the wall and whereinthe ridges are configured to cause compression of the cells based onviscoelasticity of the cells, wherein the viscoelasticity corresponds toability of the cells to pass through the compression gaps; an expansionregion, wherein the expansion region is disposed downstream from theridges; and one or more collection points, disposed downstream theexpansion region and configured to collect the cells.